National CJD Research & Surveillance Unit, Centre for Clinical Brain Sciences, University of Edinburgh, Edinburgh, Scotland, UKInstitute for Neurodegenerative Diseases, University of California, San Francisco, San Francisco, CA

Medical Research Council Centre for Regenerative Medicine, University of Edinburgh, Edinburgh, Scotland, UKRoyal (Dick) School of Veterinary Studies and The Roslin Institute, University of Edinburgh, Edinburgh, Scotland, UK

Medical Research Council Centre for Regenerative Medicine, University of Edinburgh, Edinburgh, Scotland, UKUK Dementia Research Institute, University of Edinburgh, Edinburgh, Scotland, UKCentre for Clinical Brain Sciences, University of Edinburgh, Edinburgh, Scotland, UKCentre for Brain Development and Repair, Institute for Stem Cell Biology and Regenerative Medicine, National Centre for Biological Sciences, Bangalore, India

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Article Figures & Data

Figures

Characteristics of iPSC-derived astroglial progenitor cells and astrocytes. (A) Schematic representation of differentiation of healthy donor cells to astrocyte progenitors and astrocytes. (B) Representative immunographs and quantification of nestin and vimentin coexpression in APC cultures. (C) Representative immunographs of astrocytes of all lines immunostained for GFAP and GLAST from three replicate experiments. (D) A majority of cells of all lines are GFAP-expressing cells (iPSC1, 87.8 ± 1.7%; iPSC2, 91.4 ± 1.7%; and iPSC3, 89.2 ± 0.7%), with a minor proportion expressing the neuronal marker, NeuN (iPSC1, 6.3 ± 2%; iPSC2, 5.4 ± 1.8%; and iPSC3, 6 ± 0.4%). (E) Proliferating Ki67-positive cells in iPSC1, iPSC2, and iPSC3 APC and astrocyte cultures. (F) Functional evaluation of APC and astrocyte cultures by l-glutamate uptake assay. (G) Representative immunoblots of PrPC expression in MM (iPSC1), MV (iPSC2), and VV (iPSC3) lines as APC cultures and their corresponding CNTF differentiated astrocytes from two samples each in two replicate experiments. The amount of total protein loaded was 20 µg/lane. The blots were developed with anti-PrP 3F4 antibody and then stripped and reprobed with anti–β actin antibody as a loading control. Molecular masses are in kilodaltons. (H) Maximum intensity projection of Z stacks of VV (iPSC3) astrocytes immunolabeled for PrPC (green) and cell membrane (white) without and with Triton X-100 permeabilization. (I) Summary table of all cell lines and CJD strain combinations with their respective PRNP codon 129 polymorphisms used in this study. (J) Graphic representation of live/dead (viability/cytotoxicity) assay of iPSC1, iPSC2, and iPSC3 astrocytes exposed to 1% spin-filtered vCJD, sCJD, or NBHs for 24 h and analyzed immediately (0 dpe) or after 3 d of recovery in fresh media (3 dpe). Unexposed cells served as a baseline control for each cell line. Cell count of each group is represented as percentage of total cells, and the data were acquired from 10 randomized fields from three replicate experiments, unless otherwise stated. (B–F and J) Data are plotted as mean ± SEM and analyzed by one-way ANOVA, followed by Tukey’s multicolumn comparison test; *, P < 0.05; ****, P < 0.0001; ns, not significant. (B, C, and H) Nuclei were stained with DAPI (blue). Bars, 20 µm.

Dose-dependent accumulation and subpassage of CJD prions in iPSC-derived astrocytes. (A) Immunoblot analysis of astrocytes exposed to vCJD (MM) brain homogenate at five concentrations for 24 h and assayed immediately (0 dpe) or after recovery in fresh media for 3 d (3 dpe). MM (iPSC1) astrocytes replicate PrPSc in a concentration-dependent manner. MV (iPSC2) and VV (iPSC3) astrocytes failed to replicate PrPSc at 3 dpe. The experiment was conducted twice with similar results. (B) MM (iPSC1) astrocytes exposed to 1% spin-filtered vCJD brain homogenate. Both first and second passage were analyzed at 8 dpe. In the second passage experiment, the naive MM (iPSC1) astrocytes were exposed to vCJD-infected cell homogenate diluted to match PrPSc level of original vCJD brain homogenate used for exposure of astrocytes in the first passage. n = 2, in duplicate. (C) VV (iPSC3) astrocytes exposed to 1% spin-filtered sCJD (VV2) brain homogenate (first passage) and sCJD VV2–infected astrocyte homogenate (second passage). The naive VV (iPSC3) astrocytes were exposed to a whole sCJD-infected cell homogenate (1:1), i.e., cell lysate of a single well served as an inoculum for a new well because of a lower efficiency of sCJD prion propagation. Both passages were analyzed at 8 dpe. n = 2, in triplicate. Blots were developed using anti-PrP (A and B) 3F4 and (C) HuM-P antibodies. Molecular mass is indicated in kilodaltons. (B and C) Data are plotted with mean. PK-resistant PrPSc signal values in cell lysates were normalized by the PrPSc signal value of the inoculum used in each individual experiment. (D) Representative PrP and GFAP immunolabeling in VV (iPSC3) astrocytes exposed to 1% spin-filtered sCJD (VV2) brain homogenate (first passage, middle). VV astrocytes exposed to spin-filtered cell homogenate of astrocytes propagating sCJD (VV2; second passage, right). Both were analyzed at 8 dpe. PrP immunolabeling intensity (bright green) showed an increase in cell-associated PrPSc when astrocytes of first sCJD passage were compared with PrPC of unexposed control cells (control, left), and PrPSc signal appeared more abundant in astrocytes in the second passage. n = 2, in triplicate. (E) Maximum intensity projection of Z stacks of VV (iPSC3) astrocytes immunolabeled for PrP at 8 dpe in cells exposed to sCJD (VV2) infected cell homogenate (right) and unexposed control (left). (D and E) Cells were immunolabeled with anti-PrP antibody 3F4. Nuclei were stained with DAPI (blue). Bars: (D) 20 µm; (E) 5 µm.